Spinal muscular atrophy (SMA) is a neurological disorder that results from loss of function of the anterior horn cells in the spinal cord, manifesting as progressive motor weakness, muscle wasting, and paralysis. SMA is caused by insufficient levels of the survival motor neuron (SMN) protein. The SMN locus on chromosome 5q13 contains two inverted copies of SMN called SMN1 and SMN2. Most cases of SMA harbor homozygous deletions of the SMN1 gene and retain at least one copy of SMN2. With a carrier rate of about 1 in 40, SMA is estimated to be the most frequent genetic cause of infant mortality.
SMN2 is a gene duplication of SMN1 with the same predicted amino acid coding capacity. The nucleotide sequences of SMN1 and SMN2 are nearly identical. A critical difference is a C to T transition at the +6 position in exon 7, which dramatically influences the splicing pattern in these genes. Greater than 90% of SMN1 transcripts include exon 7, while there is less than 15% exon 7 inclusion in SMN2 transcripts. This alternatively spliced product produces a truncated and unstable form of the SMN protein. Any increase in the inclusion of exon 7 in SMN2 transcripts would result in higher levels of full length SMN protein. A treatment that increases the amount of full length SMN2 mRNA should result in increased levels of SMN protein. Based on this premise, an in vivo screen that can detect increases in full-length exon 7 included SMN2 transcripts was developed.
A splicing reporter that fused SMN exons 6, 7, and 8 and their introns in frame with firefly luciferase and was expressed from a CMV promoter in C33a cells has previously been constructed. It was found that this reporter could recapitulate changes in splicing observed with over-expression of the splicing factor Tra2β. This assay was used successfully to identify compounds that increase the amount of full-length transcript produced by the SMN2 gene and SMN protein in fibroblasts isolated from an SMA patient. Another study used a SMN2 promoter based reporter to screen a library of small molecules for ability to increase SMN expression levels in NSC34 cells. This reporter measured only SMN2 specific transcription and lacked any SMN gene sequence.
It has been reported that histone deacetylase (HDAC) inhibitors such as sodium butyrate, trichostatin A (TSA), valproic acid, suberoylanilide hydroxamic acid (SAHA) and LBH589 increase SMN transcription and inclusion of exon 7. For many of these HDACs, relatively high (micromolar or millimolar) concentrations of these compounds are necessary. These activators are non-specific and will alter transcription of many genes so long-term safety has been questioned. However, type I severe SMA is fatal and short-term administration of such compounds may provide limited benefits.
A first generation splicing assay had low signal intensity, high basal expression of SMN-luciferase, and became less responsive with serial cell passage. These cells were determined to be unsuitable for high-throughput screening (HTS). The reporter system was redesigned and a more stable and reproducible assay has now been built that may be used for HTS.
Particularly, described herein is a clonal second generation SMN-luciferase reporter cell line that combines the strengths of both the promoter-based assay and a previous splicing reporter. This assay is much more robust, has lower well-to-well variation, and displays more stable luciferase expression that does not change with serial passage. It also faithfully reproduces the reported activity of an array of drug-like compounds that have been shown to increase SMN expression levels. This reporter can detect changes in SMN2 levels in response to overexpression of splicing factors such as Tra2β. This assay is a vast improvement on the previous generation of reporters and represents a valuable tool for further identification and characterization of compounds that increase expression of full-length SMN protein from the SMN2 gene.
Additionally, there is a need for new drugs to treat spinal muscular atrophy. SMN reporters can be used as tools for identifying and characterizing protein factors and chemical compounds that increase expression of full-length SMN2 transcripts. Results from HTSs to identify novel compounds that increase SMN2 expression using this cell based SMN-luciferase reporter assay are also described herein.
As described herein, the present disclosure provides compounds useful for treating or lessening the severity of spinal muscular atrophy. The present disclosure also provides methods of treating or lessening the severity of spinal muscular atrophy comprising administering to a patient susceptible to or having spinal muscular atrophy a compound or composition of the present disclosure.